Epigenetic abnormalities participate with genetic mutations to cause cancer; consequently epigenetic intervention of cancer has emerged as a promising avenue toward cancer therapy. Selective inhibition of histone deacetylases (HDACs) by small molecules often leads to a cascade of chromatin remodeling, tumor suppressor gene reactivation, apoptosis, and regression of cancer. First-in-class HDAC inhibitor on the market, Zolinza (by Merck) - a synthetic compound, was approved for the treatment of cutaneous T-cell lymphoma in late 2006. FK228 represents a small family of rare natural products with unique structural properties, potent HDAC inhibition activities, good anticancer therapeutic index, but also certain levels of undesirable cytotoxicity. Our studies of the biosynthesis of FK228 and spiruchostatins have led to the discovery of thailandepsins A and B, two new naturally produced FK228-family analogs with better HDAC inhibition activities and predicted anticancer activities currently being evaluated at the US National Cancer Institute (NCI) Developmental Therapeutics Program (DTP). Our working hypothesis is that targeted discovery from prioritized microorganisms and biosynthetic engineering of parallel metabolic pathways could lead to the acquisition of additional FK228 analogs with better anticancer therapeutic index. Thus, the overall goal of this application is to explore naturally produced and metabolically engineered HDAC inhibitors as anticancer lead compounds. To achieve this goal we will pursue the following studies: Firstly, we will characterize new genes and new pathways involved in the biosynthesis of FK228-family natural products in selected bacterial species. This study will provide important guidance for downstream engineered biosynthesis efforts. Secondly, we will discover and engineer a library of new FK228- family analogs by means of targeted bioprospecting, genome mining, simple gene deletion or insertion, complex genetic manipulation, combinatorial biosynthesis, precursor-directed mutasynthesis, de novo biosynthesis and engineering in heterologous hosts, and chemoenzymatic synthesis. Finally, we will identify new HDAC inhibitors and new anticancer lead compounds through in vitro enzyme inhibition assays and collaborative screening against the NCI's 60 cancer cell lines.